Immune system stabilizers for prevention and therapy of disorders associated with immune system disfunction

ABSTRACT

Substances that bind to alloimmune-immunogen-absorbed (AIA) sera have been found to be effective in the prevention and therapy of autoimmune disease. It is conjectured that the mechanism involves indirect stimulation of suppressor T cells resulting in stabilization of the immune system. The administration of such substances is expected to be useful in the prevention and treatment of diseases that involve a failure of the immune system to distinguish adequately between self and nonself, including autoimmune diseases and immunodeficiency diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of U.S. patent application Ser. No.07/551,262, filed Jul. 11, 1990, now abandoned.

TECHNICAL FIELD

The invention relates to the application of certain substances toprevention and therapy of conditions that reflect an immunodeficiencyinvolving a failure to adequately distinguish between self and non-self.Administration of sub-immunogenic amounts of substances that areimmunoreactive with alloimmune sera that have been absorbed against theimmunogen used in producing the alloimmune serum can prevent immunesystem disfunction, and such substances can also be used in therapy. Themechanism underlying this invention is thought to involve indirectstimulation of suppressor T cells of the immune system.

BACKGROUND ART

No consensus exists concerning the mechanisms involved in the regulationof the immune system. One view is that the immune system is a network ofcells that not only recognize foreign substances, but also recognize andregulate each other. Hoffmann et al. have developed a model of immunesystem regulation that provides the conceptual framework from which thepresent invention emerged (Hoffmann, 1975; 1978; 1980; 1981; 1982; 1988;1990; Gunther and Hoffmann, 1982, Hoffmann et al., 1988; Hoffmann andGrant, 1989; Grant et al., 1989; Hoffmann and Grant, 1990; Hoffmann etal., 1990). The model includes suppressor T cells with idiotypes thathave similarity to class II MHC in the sense that both class II MHC andthe suppressor T cell iiotypes in question have complementarity tohelper T cell idiotypes (Hoffmann, 1988; Hoffmann et al., 1988).

AIDS is an immunodeficiency disease that results in the development ofboth autoimmunity (Ziegler and Stites, 1986; Shearer, 1986; Andrieu, etal., 1986; Martinez-A , et al., 1988; Siliciano et al., 1988; Grant etal., 1990) and cancers such as Kaposi's sarcoma and lymphomas. Aunifying aspect of the occurence of both cancer and autoimmunity in AIDSmay be a failure of the immune system to distinguish properly betweenself and non-self. The possible importance of autoimmunity in AIDS isillustrated by the fact that 31 similarities have been noted betweenAIDS and systemic lupus erythematosus (Kaye, 1989). Shearer suggestedthat alloimmunity may be important in AIDS pathogenesis (Shearer, 1983),while Ziegler and Stites proposed the first autoimmunity model of AIDSpathogenesis based on network ideas (Ziegler and Stites, 1986). Hoffmannet al. have developed a model of AIDS pathogenesis based onautoimmunity, alloimmunity and network ideas (Hoffmann, 1988; Hoffmannet al.; 1988; Hoffmann and Grant, 1989; Hoffmann, Kion and Grant, 1988;Hoffmann et al., 1989; Grant et al., 1989; Hoffmann, Kion and Grant,1990; Hoffmann, 1990). This model has been called the MIAMI model ofAIDS pathogenesis, which is short for MHC-Image-Anti-MHC-Image; themodel involves synergy between MHC-image immunity and anti-MHC-imageimmunity in destabilizing the immune system. Experimental evidencesupporting such a model of pathogenesis has been recently been obtainedfor the MRL mouse (Kion and Hoffmann, 1990). The MRL mouse is a modelfor the disease systemic lupus erythematosus. Several cancers occur inAIDS, including Kaposi's sarcoma and lymphomas. According to the immunesurveillance theory of oncogenesis, cancers occur when the immune systemfails to recognize them as abnormal or "foreign", and does not eliminatethem. Hence the formation of at least some cancers may be a consequenceof a failure in self-nonself discrimination by the immune system.

A variety of conditions similar to AIDS exist in animals and may haveetiologies similar to that of AIDS (Salzman, 1986).

Stimulation of the immune system in a way that enhances and/orstabilizes its ability to distinguish between self and non-self, willprevent some failures of the immune system including AIDS and systemiclupus erythematosus.

DISCLOSURE OF THE INVENTION

The invention in one aspect is directed to specifically modify thestatus of the immune system to prevent and treat diseases in which theimmune system of a vertebrate subject fails to adequately distinguishbetween self and nonself. Such diseases include AIDS, autoimmunediseases such as systemic lupus erythematosus, and cancers associatedwith immunodeficiencies. The method involves the administration of asubstance that stabilizes the immune system network (an "immune systemstabilizer"). The immune system stabilizer of this invention is asubstance that reacts with AIA sera (alloimmune-immunogen-absorbed sera,that is, alloimmune sera absorbed with the immunogen used in producingthe sera) more strongly than with normal (non-immune) serum from thesame species. Methods of treatment and prevention are the same, exceptthat they may involve different regimens of administration of the immunesystem stabilizer.

While not intending the invention to be bound by any theory, the immunesystem stabilizer is believed to contain an antigenic component thatindirectly or directly stimulates suppressor T cells. In our networkview of the immune system, at least some suppressor T cells are believedto stabilize the immune system network through their interactions withhelper T cells (see Hoffmann 1988; Hoffmann et al., 1988).

Substances that react with alloimmune-immunogen-absorbed ("AIA") serainclude gp120 of the human immunodeficiency virus ("HIV") and p24 ofHIV. As partial proof of principle, both of these substances have beenfound to inhibit the progression of autoimmune disease in a murine modelof autoimmunity, namely the MRL-1pr/1pr mouse.

In another respect, the invention is directed to pharmaceuticalcompositions suitable for the conduct of the method of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows that gp120 of HIV binds more strongly to murine AIA(alloimmune-immunogen-absorbed) sera than to normal mouse serum. FIG. 1Bshows that p24 of HIV binds more strongly to murine AIA(alloimmune-immunogen-absorbed) sera than to normal mouse serum.

FIG. 2 shows the level of various diagnostic antibodies (anti-DNA,anti-collagen and anti-gp120) in a strain of mice that is a model forautoimmunity following injections of gp120 and p24 from HIV, with thelevels for similar mice injected with phosphate buffered saline as anegative control. FIG. 2A shows that the level of anti-gp120 in thesemice is reduced (compared with the level in controls) as a result of theinjections; FIG. 2B shows that the level of anti-collagen is reduced asa result of such injections, and FIG. 2C shows that the level ofanti-DNA antibodies in these mice is reduced as a result of suchinjections.

FIG. 3 shows survival data (as of mid-Dec., 1990) for mice used in theexperiment of FIG. 2. The "experimental group" data are pooled data forthe p24 and gp120-injected mice; four mice received p24 of HIV, and fourmice received gp120 of HIV. The control mice received either nothing (15mice) or phosphate buffered saline (4 mice from group 1 of the FIG. 2experiment). At the time of submission, the p24-injected mice are allalive (4/4), and 3 of the 4 gp120-injected mice are alive, in contrastto only about 20% of the control mice being alive at the correspondingtime point.

MODES OF CARRYING OUT THE INVENTION

The invention is directed to a method to modify the immune system of asubject and thus to (a) prevent diseases involving a failure of theimmune system to adequately distinguish between self and nonself, and(b) provide a therapy for such conditions. This is done byadministration(s) of a substance(s) that reacts with AIA(alloimmune-immunogen-absorbed) sera, in a way that does not induceimmunity to the said substance, but that is found empirically tostabilize the system in a comparatively healthy state.

Preparation of AIA Alloimmune-immunogen-absorbed sera or AIA is preparedfrom the sera of a vertebrate that has been made hyperimmune withrespect to allogeneic lymphocytes as detailed in Hoffmann et al., 1986,incorporated herein by reference. Alloimmune murine sera are produced byfirst immunizing mice with 6 weekly injections of 5×10⁷ allogeneiclymphoid cells (lymph node, spleen and thymus) in phosphate bufferedsaline, pH 7.2 (PBS). The mice are first bled 7 days after the sixthinjection, and thereafter they are injected with 10⁷ allogeneic lymphoidcells 7 days prior to subsequent bleedings at 2 week intervals. Theantisera are pooled, heat inactivated at 56° C. for 30 minutes andstored at -20° C. until use. AIA is produced by absorbing the alloimmuneserum by 5 serial absorptions against 2×10⁹ glutaraldehyde-fixedlymphocytes (of the strain used in the immunizations) per ml. of serum.Each absorption is done at room temperature for 1 hour with occasionalmixing. Such absorptions remove all detectable cytotoxic activityagainst the immunizing cells in the serum.

Human AIA can be obtained, for example, from the alloimmune serum of amonogamous, multiparous mother with a high titre of immunity to herhusband's cells. The absorption against the husband's lymphocytes isdone as described above for immune murine serum.

Either murine AIA or human AIA can be used to screen candidate antigens.Since murine AIA reacts with p24 and gp120 of the human virus HIV, andsince these human viral components provide protection and therapy for amurine autoimmune disease, it is possible that murine AIA and human AIAmay be approximately equally suitable for screening for AIA antigens foruse in humans.

In addition, our idiotypic network model of AIDS pathogenesis (Hoffmann,Kion and Grant, 1990) suggests that HIV components have determinantsthat are cross-reactive with MHC-image determinants on the T cellreceptors of certain suppressor T cells. There may be considerablecross-reactivity between MHC-image determinants in various species.Gp120 and p24 of HIV are examples of substances that are obtained from ahuman virus yet bind to murine AIA, and that have been shown to work inprevention and therapy of murine autoimmunity (see below). This suggeststhat there is a considerable amount of interspecies cross-reactivity inAIA, and murine AIA may well be a suitable reagent for screening forhuman immune system stabilizers.

Screening Candidate Antigens Against AIA Serum

An ELISA assay is conveniently used. ELISA plates are coated with thesubstance to be tested, the plates blocked with 5% casein, thenincubated with murine AIA serum. Bound antibodies are detected usingbiotinylated goat anti-mouse IgG and avidin-alkaline phosphatase. Ananalogous assay is also conducted but substituting normal non-immuneserum from the same species as the AIA serum for the AIA serum in theassay. Substances which react more strongly with the AIA serum than withthe normal serum are useful antigens. Other protocols and detectionsystems can be used as is understood in the art.

Selection of Candidate Substances For Binding To AIA Serum

Suitable candidates are substances for which there is some reason tobelieve that they may have shapes that mimic the shape of majorhistocompatability complex antigens For example, there is much evidencesuggesting that some HIV components have shapes that mimic MHC class IIantigens (discussed in Hoffmann et al., 1990).

Illustrative AIA-reactive substances include gp120 and p24 of HIV (seebelow). They may also include gp41 and Nef of HIV, since gp41 has beenreported to have serological cross-reactivity with class II MHC (Goldinget al., 1989), and Nef has some sequence homology with class II MHC(Vega et al., 1990).

Another class of AIA-reactive substances are some of the antibodiespresent in alloimmune sera, or monoclonal antibodies derived fromalloimmune vertebrates. Antibodies contained within one AIA often reactwith antibodies in another AIA (Kion and Hoffmann, unpublished). Forexample, if a mouse of strain A is made immune to lymphocytes from miceof a strain B (that has a different MHC haplotype), and if a mouse ofstrain B is similarly made immune to lymphocytes from mice of strain A,each of the resulting AIA sera contains antibodies that react with theother AIA. This is due to the fact that AIA typically contain bothMHC-image and anti-MHC-image antibody activities, and MHC-imageantibodies in one AIA typically react with anti-MHC-image antibodies inanother AIA.

Still another class of AIA-reactive substances ar anti-I-J antibodies.These are a class of murine antibodies that are present in somealloimmune sera and that react mainly with suppressor T cells. It hasbeen suggested that anti-I-J antibodies are anti-MHC-image antibodies(Hoffmann, 1988; Hoffmann et al., 1988). We, find that monoclonalanti-I-J antibodies bind to AIA (Kion and Hoffmann, unpublished), andthey may be effective reagents in the context of this invention.

Utility and Administration

The antigens immunoreactive with AIA serum are immune system stabilizersand are administered in subimmunogenic doses to suitable subjects As thedosage levels are subimmunogenic, the risk factors in administeringthese materials as prophylactics to the general population are very low.Particularly preferred subjects are those in the more elderlypopulation, as the ability of the immune system to discriminate betweenself and non-self deteriorates with age. This is evidenced by anincreasing amount of anti-self antibodies in the immune responses ofolder people and an increase with age and the prevalence of autoimmunephenomena in cancers.

The immune system stabilizers of the invention are also usefultherapeutic agents in treatment of subjects with known autoimmuneconditions. Thus, the invention protocol can be used to treat variousconditions affecting the immune system, such as acquiredimmunodeficiency syndrome (AIDS) and systemic lupus erythematosus (SLE)and other autoimmune diseases such as myasthenia gravis, rheumatoidarthritis, and the like. The dosage levels are at a subimmunogenic leveland are thus expected to be in the range of 10⁴ -10⁶ fold less thanamounts required for an immunogenic dose. The appropriate dosage levelsfor a particular subject can be determined by reference to a suitableanimal model. For example, in determining dosage levels suitable for thetreatment of human diseases such as AIDS and SLE, reference can be hadto animal models to determine the relative amounts of antigen to beadministered.

Administration is by any systemic route, and is typically by injection,such as intramuscular, subcutaneous, intraperitoneal, or intravenousinjection. Suitable formulations for injection are understood in the artand include solutions or suspensions of the antigen in a compatibleaqueous medium such as Hank's solution or Ringer's solution. Systemicadministration can also be effected by transmucosal or transdermaldelivery using appropriate penetrants and absorption mediators, such asbile salts, fusidates and various detergents. Transmucosaladministration is typically effected by nasal sprays, suppositories, orintroduction into the lungs. In addition, when properly formulated, theimmune system stabilizers of the invention may be administered orally.For oral administration, however, the formulation must provide a meansfor transport of the drug to the bloodstream.

Suitable formulations for a particular mode of administration aredesigned using techniques within ordinary skill of the art. Suchformulations are cataloged, for example, in "Remington's PharmaceuticalSciences," latest edition, Mack Publishing Co., Easton, Pa. All of theformulations may contain various excipients which are benign andpharmaceutically acceptable, including various carriers, buffers,stabilizing agents, wetting agents and the like.

The particular formulation, dosage level and mode of administrationchosen will depend on the nature of the condition to which the immunestabilization is directed, on the general health and nature of thesubject, and on the judgment of the practitioner. Suitable subjectsinclude not only humans, but animals; the immune system stabilizers mayalso be appropriately formulated for veterinary use.

EXAMPLES

The following examples illustrate but do not limit the invention.

Example 1

Gp120 and p24 of HIV bind to murine AIA sera. Alloimmune sera wereraised in Balb/c and C57BL/6 (B6) mice by repeated reciprocalimmunizations using B6 and Balb/c lymphoid cells. The sera of theresulting alloimmune mice were then tested for the presence ofantibodies immunoreactive with gp120 and p24 of HIV. Antibodiesimmunoreactive with both of these viral components were detected inalloimmune mice but not in non-immune controls. These results are shownin FIGS. 1A and 1B for anti-gp120 and anti-p24 respectively. Thetriangles show the results for the non-immune control sera, the opensquares and full squares are for B6 anti-Balb/c and Balb/c anti-B6immune sera respectively, and the circles are foralloimmune-immunogen-absorbed sera, namely B6 anti-Balb/c absorbed withBalb/c (open circles) and Balb/c anti-B6 absorbed with B6 cells (fullcircles). It is apparent that absorption with the immunogen (whichremoves the cytotoxic activity of the sera) does not remove theanti-gp120 or anti-p24 immuno-reactivity.

Example 2

Prevention of Autoimmunity in MRL-1pr/1pr Mice: MRL-1pr/1pr is a mousestrain that develops a lupus-like autoimmune disease. The AIA-bindingsubstances gp120 and p24 of HIV inhibit the formation of autoantibodiesin MRL-1pr/1pr mice. MRL-1pr/1pr mice were immunized intraperitoneallywith the following antigens at ages 3 weeks and 4 weeks. (PBS isphosphate buffered saline).

Group 1: 0.2 ml PBS alone

Group 2: 10 ng of anti-I-A^(k) monoclonal antibody in 0.2 ml PBS.

Group 3: 10 ng of gp120 of HIV in 0.2 ml PBS.

Group 4: 10 ng of p24 of HIV in 0.2 ml PBS.

All mice were bled at 4 weeks after the second injection, and the serawere obtained as individual samples, heat inactivated at 56° C. for 30minutes.

FIG. 2 shows the level of various diagnostic antibodies in a strain ofmice that is a model for autoimmunity following injections of gp120 andp24 from HIV, with the levels for similar mice injected with phosphatebuffered saline as a negative control. FIG. 2A shows that the level ofanti-gp120 in these mice is reduced (compared with the level incontrols) as the result of the injections; FIG. 2B shows that the levelof anti-collagen is reduced as a result of such injections, and FIG. 2Cshows that the level of anti-DNA antibodies in these mice is reduced asa result of such injections.

Example 3

The AIA-binding substances gp120 and p24 of HIV enhance longevity.MRL-1pr/1pr mice injected with AIA-binding material only at 3 weeks and4 weeks of age (the mice used in the preceding experiment) are beingmonitored for longevity. This experiment is still in progress (Dec. 13,1990), but it is already clear that especially mice in the groups 3 and4 of the above experiment are living longer than control mice. Mostdramatically, in the group of four that were injected with p24 (group 4above) , 4 of 4 are alive and well at day 211, when about 80% of thecontrol mice are dead. In the gp120 group (group 3 above) 3 of 4 miceare alive at 211 days. In FIG. 3, the "experimental group" is pooleddata for the p24 and gp120 injected groups of mice. The "control group"of FIG. 3 shows data for the PBS-injected group (group 1 above) pooledwith a larger group of 15 MRL-1pr/1pr mice that received no injections.This combined group of mice have a median life span of 178 days, andonly 4 of 19 were alive at 205 days.

Example 4

Therapy for autoimmunity in MRL-1pr/1pr mice. Three month oldMRL-1pr/1pr mice exhibit a significant amount of autoimmunity. Weeklyinjections of 10 ng of gp120 of HIV beginning at age 3 months have beenfound to markedly inhibit the development of lymphadenopathy in thesemice (relative to phosphate buffered saline injected controls), andafter eleven weeks of these injections these mice appear much healthierthan the controls.

References

Andrieu, J. M., Even, P. and Venet, A. (1986) AIDS Research 2, 163-174.

Calabrese, L. H. (1988) Clin. Lab. Med. 8, 269.

Golding, H., Shearer, G. M., Hillman, K., Lucas, P., Manishewitz, J.,Zajac, R. A., Clerici, M., Gress, R. E., Boswell, R. N., and Golding, B.(1989) J. Clin. Invest 83, 1430-1435.

Grant, M. D., Weaver, M. S., Tsoukas, C. and Hoffmann, G. W. (1990): J.Immunol., 144, 1241-1250.

Grant, M., Kion, T. A., Forsyth, R. B., and Hoffmann, G. W. (1989) in J.G. Kaplan and D. R. Green, eds., A. R. Liss, Inc., New York, pp.481-484.

Gunther, N. and Hoffmann, G. W. (1982), Journal of Theoretical Biology,94, 815-855.

Habeshaw J. A. & Dagleish, A. (1989) J. AIDS, 2, 457-468.

Hoffmann, G. W. (1975) European Journal of Immunology, 5, 638-647(1975).

Hoffmann, G. W. (1978) in "Theoretical Immunology" G. I. Bell, A. S.Perelson and G. H. Pimbley (eds.) Marcel Dekker, N.Y., pp. 571-602.

Hoffmann, G. W. (1980) Contemp. Top. Immunobiol. 11, 185-226.

Hoffmann, G. W. (1981) in The Immune System, Festschrift in Honour ofNiels Kaj Jerne, on the Occasion of his 70th Birthday (I. Lefkovits andC. Steinberg, Eds.) Karger, Basel, vol. I, pp. 28-34. Hoffmann, G. W.(1982) in Regulation of Immune Response Dynamics, C. DeLisi and J.Hiernaux (Eds.), CRC Press, pp. 137-162.

Hoffmann, G. W. (1988) in "The Semiotics of cellular Communication inthe Immune System" (E. Sercarz, F. Celada, N. A. Mitchison and T. Tada,Eds.) Springer-Verlag, New York, pp. 257-271.

Hoffmann, G. W. (1990) Res. in Immunol., in press.

Hoffmann, G. W. and Grant, M. D. (1989) in "Mathematical and StatisticalApproaches to AIDS Epidemiology," C. Castillo-Chavez, Ed., Lecture Notesin Biomathematics, vol. 83, Springer-Verlag, 1989, pp. 386-405.

Hoffmann, G. W. and Grant, M. D. (1990) in "Idiotype Networks in Biologyand Medicine", A. D. M. E. Osterhaus and F. G. C. M. UytdeHaag, Eds.,Elsevier, Amsterdam, pp. 295-299.

Hoffmann, G. W. and Tufaro, F. (1989) Immunol. Letters, 22, 83-90.

Hoffmann, G. W., Cooper-Willis, A. and Chow, M. (1986) J. Immunol. 137,61-68.

Hoffmann, G. W., Grant M. and Kion, T. A. (1988) Canadian Research, 21,No. 6, pp. 16-23.

Hoffmann, G. W., Kion, T. A. and Grant, M. D. (1990) "An IdiotypicNetwork Model of AIDS Immunopathogenesis" Proc. Nat. Acad. Sci. (USA),in press.

Hoffmann, G. W., Kion, T. A., Forsyth, R. B., Soga, K. G. andCooper-Willis, A. (1988) in "Theoretical Immunology, Part Two", A. S.Perelson, Ed., Santa Fe Institute Series "Studies in the Science ofComplexity", Addison Wesley Publishing Company, 291-319.

Kaye, B. R., (1989) Ann. Int. Med. 111, 158-167. Kion, T. A. andHoffmann, G. W. (1990), submitted for publication.

Lanzavecchia, A. (1989) Research in Immunol. 140, 99-103.

Martinez-A, C., Marcos, M. A. R., de la Hera, A., Marquez, C., Alonso,J. M., Toribio, M. L. and Coutinho, A. (1988) Lancet, Feb. 27, 1988.

Morrow, W. J. W., Isenberg, D. A., Sobol, R. E., Stricker, R. B. andKieber-Emmons, T. Clin. Immunol. & Immunopath. in press.

Salzman, L. A. (1986) "Animal Models of Retrovirus Infection and theirRelationship to AIDS." (L. A. Salzman, editor) Academic Press.

Shearer, G. M. (1983) N. Engl. J. Med. 308, 223-224.

Shearer, G. M. (1986) Mount Sinai J. Med. 53, 609-615.

Siliciano, R. F., Lawton, T., C. Knall, C., Karr, R. W. Bermann, P.,Gregory, T. & Reinherz, E. L. (1988) Cell 54:561.

Vega, M. A., Guigo, R. and Smith, T. F. (1990) Nature, 345, 26.

Ziegler J. L. and Stites, D. P. (1986) Clin Immunol. and Immunopath. 41,305-313.

I claim:
 1. A method for treating or inhibiting the development ofsymptoms of an autoimmune disease in a subject, which method comprisesadministering to the subject a subimmunogenic amount of an antigen moreimmunoreactive with alloimmune-immunogen-absorbed (AIA) serum ascompared to nonimmune serum of the same species as the AIA serum.
 2. Themethod of claim 1 wherein the AIA serum is murine.
 3. The method ofclaim 1 wherein the AIA serum is human.
 4. The method of claim 1 whereinthe subject is human.
 5. The method of claim 1 wherein the subject is anonhuman vertebrate.
 6. The method of claim 1 wherein the antigencomprises gp41 from HIV.
 7. A method to activate T-suppressor cells in asubject susceptible to or suffering from symptoms of an autoimmunedisease, which method comprises administering to a subject in needthereof a subimmunogenic amount of an antigen more immunoreactive withalloimmune-immunogen-absorbed (AIA) serum as compared to nonimmune serumof the same species as the AIA serum.
 8. The method of claim 1, whereinthe autoimmune disease is systemic lupus erythematosus.
 9. The method ofclaim 7, wherein the autoimmune disease is systemic lupus erythematosus.10. A method to inhibit the production of autoantibodies in a subjectsusceptible to or suffering from an autoimmune disease which methodcomprises administering to the subject a subimmunogenic amount of anantigen which is more immunoreactive with alloimmune-immunogen-absorbed(AIA) serum as compared to nonimmune serum of the same species as theAIA serum.
 11. The method of claim 10 wherein the autoantibodies areanti-collagen.
 12. The method of claim 10 wherein the autoantibodies areanti-DNA.